Somatostatin (SS), a tetradecapeptide discovered by Brazeau et al., has been shown to have potent inhibitory effects on various secretory processes in tissues such as pituitary, pancreas and gastrointestinal tract. SS also acts as a neuromodulator in the central nervous system. These biological effects of SS, all inhibitory in nature, are elicited through a series of G protein coupled receptors, of which five different subtypes have been characterized (SSTR1-SSTR5) (Reubi J C, et al., Cancer Res 47: 551-558, Reisine T, et al., Endocrine Review 16: 427-442, Lamberts S W, et al., Endocr Rev 12: 450-482, 4 Patel Y C, 1999 Front Neuroendocrinology 20: 157-198). These five subtypes have similar affinities for the endogenous SS ligands but have differing distribution in various tissues. Somatostatin binds to the five distinct receptor (SSTR) subtypes with relatively high and equal affinity for each subtype.
There is evidence that SS regulates cell proliferation by arresting cell growth via SSTR1, 2, 4, and 5 subtypes (Buscail L, et al., 1995 Proc Natl Acad Sci USA 92: 1580-1584; Buscail L, et al., 1994 Proc Natl Acad Sci USA 91: 2315-2319; Florio T, et al., 1999 Mol Endocrinol 13: 24-37; Sharma K, et al., 1999 Mol Endocrinol 13: 82-90), or by inducing apoptosis via SSTR3 subtype (Sharma K, et al., 1996 Mol Endocrinol 10: 1688-1696). SS and various analogues have been shown to inhibit normal and neoplastic cell proliferation in vitro and vivo (Lamberts S W, et al., Endocr Rev 12: 450-482) via specific SS receptors (SSTR's) (Patel Y C, 1999 Front Neuroendocrinology 20: 157-198) and possibly different postreceptor actions (Weckbecker G, et al., Pharmacol Ther 60: 245-264; Bell G I, Reisine T 1993 Trends Neurosci 16: 34-38; Patel Y C, et al., Biochem Biophys Res Commun 198: 605-612; Law S F, et al., Cell Signal 7:1-8). In addition, there is evidence that distinct SSTR subtypes are expressed in normal and neoplastic human tissues (Virgolini I, et al., Eur J Clin Invest 27: 645-647), conferring different tissue affinities for various SS analogues and variable clinical response to their therapeutic effects.
Binding to the different types of somatostatin receptor subtypes has been associated with the treatment of various conditions and/or diseases. For example, the inhibition of growth hormone has been attributed to the somatostatin type-2 receptor (“SSTR2”) (Raynor, et al., Molecular Pharmacol. 43:838 (1993); Lloyd, et al., Am. J. Physiol. 268:G102 (1995)) while the inhibition of insulin has been attributed to the somatostatin type-5 receptor (“SSTR5”) (Coy, et al. 197:366-371 (1993)). Activation of types 2 and 5 have been associated with growth hormone suppression and more particularly GH secreting adenomas (Acromegaly) and TSH secreting adenomas. Activation of type 2 but not type 5 has been associated with treating prolactin secreting adenomas. Other indications associated with activation of the somatostatin receptor subtypes include inhibition of insulin and/or glucagon for treating diabetes mellitus, angiopathy, proliferative retinopathy, dawn phenomenon and nephropathy; inhibition of gastric acid secretion and more particularly peptic ulcers, enterocutaneous and pancreaticocutaneous fistula, irritable bowel syndrome, Dumping syndrome, watery diarrhea syndrome, AIDS related diarrhea, chemotherapy-induced diarrhea, acute or chronic pancreatitis and gastrointestinal hormone secreting tumors; treatment of cancer such as hepatoma; inhibition of angiogenesis; treatment of inflammatory disorders such as arthritis; retinopathy; chronic allograft rejection; angioplasty; preventing graft vessel and gastrointestinal bleeding. It is preferred to have an analog which is selective for the specific somatostatin receptor subtype or subtypes responsible for the desired biological response, thus, reducing interaction with other receptor subtypes which could lead to undesirable side effects.
Somatostatin (SS) and its receptors (SSTR1 to SSTR5) are expressed in normal human parafollicular C cells and medullary thyroid carcinoma (MTC) cells. MTC is a tumor originating from thyroid parafollicular C cells that produces calcitonin (CT), somatostatin, as well as several other peptides (Moreau J P, et al., Metabolism 45 (8 Suppl 1): 24-26). Recently, Mato et al. showed that SS and SSTR's are expressed in human MTC (Mato E, et al., J Clin Endocrinol Metab 83: 2417-2420). It has been documented that SS and its analogues induce a decrease in plasma CT levels and a symptomatic improvement in MTC patients. However, until now the antiproliferative activity of SS analogues on tumor cells had not been clearly demonstrated (Mahler C, et al., Clin Endocrinol 33: 261-9; Lupoli G, et al., Cancer 78: 1114-8; Smid W M, et al., Neth J Med 40: 240-243). Thus, development and assessment of SSTR subtype analogues selective on MTC cell growth provides a useful tool for clinical application. Until now, no data concerning specific SSTR subtype involvement in MTC cell growth regulation have been reported.
The present invention relates to the discovery that the human MTC cell line TT, which displays MTC cell characteristics (Zabel M, et al., 1992 Histochemistry 102: 323-327, 2 Gagel R F, et al., 1986 Endocrinology 118: 1643-1651, Liu J L, et al., 1995 Endocrinology 136: 2389-2396) and which stably expresses all the SSTR subtypes, responds to SSTR2 and SSTR5 activation by subtype selective agonists with two different patterns in terms of [3H]thy incorporation and cell number. SSTR2 preferential agonists significantly suppress [3H]thy incorporation, i.e., inhibit DNA synthesis, and reduce cell proliferation. SSTR5 selective agonists significantly increase [3H]thy incorporation in TT cells, i.e., increase DNA synthesis, but alone fail to influence cell proliferation. Further, SSTR2 antagonists counteract the action of SSTR2 preferential agonists on TT cells. Further still, increasing concentrations of an SSTR5 selective agonist dose-dependently prevents the suppression of TT cell [3H]thy incorporation and proliferation produced by an SSTR2 preferential agonist, and vice versa, showing an antagonism between such agonists.
Hetero- and homodimeric interactions between subtypes of the opiate (Jordan B A, et al., 1999 Nature 399:697-700.) and SS (Rocheville M, et al., 2000 J. Biol. Chem. 275:7862-7869) receptor families have been recently demonstrated. Studies in cultured pituitary adenoma cells have demonstrated that SSTR subtype 2 and 5 act synergistically in the suppression of growth hormone and prolactin secretion (Shimon I, et al., 1997 J. Clinical Invest. 100:2386-2392, Jaquet P, et al., 2000 J Clin Endocrinol Metab. 85:781-792). The finding that SSTR5 activation reduces the antiproliferative activity mediated by SSTR2 differs from results in other tissues (Patel Y C, 1999 Front Neuroendocrinology 20: 157-198, Buscail L, et al., 1995 Proc Natl Acad Sci USA 92: 1580-1584, Buscail L, et al., 1994 Proc Natl Acad Sci USA 91: 2315-2319, Sharma K, et al., 1996 Mol Endocrinol 10: 1688-1696). This is the first demonstration that SSTR subtypes 2 and 5 can act antagonistically in regulating cell growth.
Thus, SSTR2 and SSTR5 preferential agonists exert differential effects on proliferation of human medullary thyroid TT cell line in vitro, according to their specific SSTR selectivity. Proliferation of the TT cell line can be reduced by SSTR2 selective agonists, but not by SSTR5 agonists, and an SSTR5 agonist can prevent SSTR2 mediated antiproliferative effects. The key inhibitory role of SSTR2 on MTC cell proliferation demonstrates that analogues with enhanced SSTR2 affinity and selectivity versus SSTR5 would be useful as antiproliferative agents in MTC treatment.